This invention relates to compounds structurally similar to the tubulysins, conjugates thereof with a ligand, methods for making and using such compounds and conjugates, and compositions comprising such compounds and conjugates.
The tubulysins are cytotoxins originally isolated from cultures of the myxobacteria Archangium gephyra or Angiococcus disciformis, with each organism producing a different mixture of tubulysins (Sasse et al. 2000; Reichenbach et al. 1998). Their crystal structure and biosynthetic pathway have been elucidated (Steinmetz et al. 2004) and their biosynthesis genes have been sequenced (Hoefle et al. 2006b). Pretubulysin, a biosynthetic precursor of the tubulysins, also has been shown to possess some activity (Ullrich et al. 2009). (Full citations for the documents cited herein by first author or inventor and year are listed at the end of this specification.)
The tubulysins belong to a group of naturally occurring antimitotic polypeptides and depsipeptides that includes the phomopsins, the dolastatins, and the cryptophycins (Hamel 2002). Antimitotic agents other than polypeptides or depsipeptides also exist, for example paclitaxel, the maytansines, and the epothilones. During mitosis, a cell's microtubules reorganize to form the mitotic spindle, a process requiring the rapid assembly and disassembly of the microtubule constituent proteins α- and β-tubulin. Antimitotic agents block this process and prevent a cell from undergoing mitosis. At the molecular level the exact blockage mechanism may differ from one anti-mitotic agent to another. The tubulysins prevent the assembly of the tubulins into microtubules, causing the affected cells to accumulate in the G2/M phase and undergo apoptosis (Khalil et al. 2006). Paclitaxel effects the same end result by binding to microtubules and preventing their disassembly.
The tubulysins have a tetrapeptidyl scaffold constructed from one proteinogenic and three non-proteinogenic amino acid subunits as shown in formula (A): N-methylpipecolinic acid (Mep), isoleucine (Ile), tubuvaline (Tuv), and either tubuphenylalanine (Tup, R′ equals H) or tubutyrosine (Tut, R′ equals OH). Among the better-known naturally occurring tubulysins (designated A, B, etc.), the sites of structural variation are at residues R′, R″ and R′″ of formula (A), as shown in Table 1:

TABLE 1Naturally Occurring TubulysinsTubulysinR′R″R′″AOHOC(═O)MeCH2OC(═O)i-BuBOHOC(═O)MeCH2OC(═O)n-PrCOHOC(═O)MeCH2OC(═O)EtDHOC(═O)MeCH2OC(═O)i-BuEHOC(═O)MeCH2OC(═O)n-PrFHOC(═O)MeCH2OC(═O)EtGOHOC(═O)MeCH2OC(═O)CH═CH2HHOC(═O)MeCH2OC(═O)MeIOHOC(═O)MeCH2OC(═O)MeUHOC(═O)MeHVHOHHYOHOC(═O)MeHZOHOHHPretubulysinHHMe
Additionally, other naturally occurring tubulysins have been identified (Chai et al. 2010).
Kaur et al. 2006 studied the antiproliferative properties of tubulysin A and found that it was more potent than other antimitotic agents such as paclitaxel and vinblastine and was active in xenograft assays against a variety of cancer cell lines. Further, tubulysin A induced apoptosis in cancer cells but not normal cells and showed significant potential anti-angiogenic properties in in vitro assays. The antimitotic properties of other tubulysins also have been evaluated and generally have been found to compare favorably against those of non-tubulysin antimitotic agents (see, e.g., Balasubramanian et al. 2009; Steinmetz et al. 2004; Wipf et al. 2004). For these reasons, there is considerable interest in the tubulysins as anti-cancer agents (see, e.g., Domling et al. 2005c; Hamel 2002).
Numerous publications describe efforts directed at the synthesis of tubulysins, including: Balasubramanian et al. 2009; Domling et al. 2006; Hoefle et al. 2003; Neri et al. 2006; Peltier et al. 2006; Sani et al. 2007; Sasse et al. 2007; Shankar et al. 2009; Shibue et al. 2009 and 2010; and Wipf et al. 2004. Other publications describe structure-activity relationship (SAR) studies, via the preparation and evaluation of tubulysin analogs or derivatives: Balasubramanian et al. 2008 and 2009; Chai et al. 2011; Domling 2006; Domling et al. 2005a; Ellman et al. 2013; Hoefle et al. 2001 & 2006a; Pando et al. 2011; Patterson et al. 2007 & 2008; Richter 2012a, 2012b, and 2012c; Shankar et al. 2013; Shibue et al. 2011; Sreejith et al. 2011; Vlahov et al. 2010a; Wang et al. 2007; Wipf et al. 2007 and 2010; and Zanda et al. 2013. The SAR studies mainly explored structural variations in the Mep ring, residues R″ and R′″ of the Tuv subunit, and the aromatic ring or aliphatic carbon chain of the Tup/Tut subunit.
Domling et al. 2005 disclose conjugates of tubulysins with a partner molecule generically described as a polymer or a biomolecule, but with examples limited to polyethylene glycol (PEG) as the partner molecule. Cheng et al. 2011 also disclose tubulysin analogs adapted for use in conjugates. Other documents disclosing conjugates of tubulysins are Boyd et al. 2008 and 2010; Jackson et al. 2013; Vlahov et al. 2008a, 2008b and 2010b; Leamon et al. 2008 and 2010; Reddy et al. 2009; and Low et al. 2010. Leung et al. 2002 disclose polyanionic polypeptides that can be conjugated to drugs (including tubulysins) to improve their bioactivity and water solubility.
Davis et al. 2008 and Schluep et al. 2009 disclose cyclodextrin based formulations in which tubulysins are covalently attached to a cyclodextrin via a hydrazide-disulfide linker moiety bonded to the Tup/Tut carboxyl group.
The deacetylation of the Tuv subunit (i.e., R″ in formula (A) is hydroxyl instead of acetyl) reportedly leads to loss of biological activity (Domling et al. 2006). In a study of tubulysins U and V, which differ in the former being acetylated and the latter being deacetylated, tubulysin V was reported to be less potent by about 200× to 600×, depending on the assay (Balasubramanian et al. 2009). Because an acetate group is susceptible to hydrolysis, deacetylation at the R″ position is a concern, as a potential instability center leading to loss of activity, for the development of tubulysin analogs for pharmaceutical applications.